TransAM® MAPK Family

TransAM® Kits are DNA-binding ELISAs that facilitate the study of transcription factor activation in mammalian tissue and cell extracts. Assays are available for over 40 different targets (see the list at right). Each kit includes a 96-stripwell plate in which multiple copies of a specific double-stranded oligonucleotide have been immobilized. When nuclear or whole-cell extract is added, activated transcription factor of interest binds the oligonucleotide at its consensus binding site and is quantified using the included antibody, which is specific for the bound, active form of the transcription factor being studied. For complete details, click the TransAM® Method tab below.

TransAM® MAPK Family Transcription Factor ELISA Kits

TransAM MAPK Family Kits provide everything needed to study transcription factors activated by Mitogen-Activated Protein Kinase (MAPK) signal transduction pathways, including a positive control extract. The MAPK Family Kit contains multiple binding sites in each well and includes antibodies specific for the active forms of the transcription factors: phosphorylated ATF-2 (Thr71), phosphorylated c-Jun (Ser73), c-Myc, MEF2 and STAT1α, all of which are regulated by MAP kinase cascades and are key markers in cell signaling. In addition to the TransAM MAPK Family Kit, individual kits for studying ATF-2, c-Jun, c-Myc and MEF2 are also available. The phosphorylated ATF-2, MEF2 and STAT1α antibodies can be used with human extracts, while the phosphorylated c-Jun and c-Myc antibodies can be detected in human and mouse extracts. Recombinant c-Fos, Recombinant c-Jun and Recombinant c-Myc proteins are available separately to generate an optional protein standard curve in the TransAM Transcription Factor ELISA kits. Click the MAPK Info tab below for data and more information; kit manuals can be downloaded under the Documents tab.

MAPK Info

The activation of Mitogen-Activated Protein Kinase (MAPK) signal transduction pathways is responsible for the phosphorylation of transcription factors by the terminal kinase in these cascades. MAPK pathways play major roles in converting mitogenic and stress stimuli into nuclear responses. At least three parallel MAPK pathways exist in humans. The extracellular signal-regulated protein kinase (ERK) pathway primarily transmits mitogenic and differentiation stimuli, while the c-Jun N-terminal kinase (JNK) and p38 pathways predominantly transmit stress and cytokine stimuli. ATF-2 (activating transcription factor 2, also known as CREB2 or cAMP responsive element binding protein 2) is a member of the ATF/CREB transcription factor family that binds to the cAMP response element (CRE). ATF-2 is activated by three MAPK pathways, ERK1/2, JNK and p38. Phosphorylation of Thr71 by MAPK14 or JNK increases transcriptional activity. MEF2, a member of the MADS box family, is mainly involved in muscle differentiation and is activated by both the p38 and ERK5 pathways. c-Myc is a cellular homolog of the avian retroviral v-myc oncogene and belongs to the Myc family, which also includes N-myc and L-myc. Translocations and deregulation of c-Myc have been implicated in cancers such as hematopoietic tumors, leukemias and lymphomas. c-Myc is activated by the MAP Kinase ERK1/2. c-Jun is a member of the activator protein-1 (AP-1) family and is activated by both ERK1/2 and JNK pathways. Jun proteins can also bind to form transcriptionally active heterodimers with CREB/ATF members. STAT1α is involved in the activation of IFNα and γ genes, and is activated by both the p38 and JNK pathways.

Figure 1: TransAM profiling of MAPK-regulated transcription factors.

The TransAM MAPK Family Kit was used to assay the activation levels of ATF-2, c-Myc, c-Jun, STAT1α and MEF2 using nuclear extracts prepared from either HeLa (TPA stimulated), Jurkat (unstimulated), K-562 (TPA stimulated) or U-937 (TPA + IFNγ treated) cells.

The TransAM® transcription factor ELISA advantage

Historically, transcription factor studies have been conducted using gelshift, Western blot and reporter plasmid transfections, which are time-consuming, do not allow for high-throughput and provide only semi-quantitative results. TransAM assays are up to 100 times more sensitive than gelshift techniques, and can be completed in less than 5 hours. Because TransAM is an ELISA-based assay*, there is no radioactivity, and the high-throughput stripwell format enables simultaneous screening of 1-96 samples. Inconsistencies due to variable reporter plasmid transfections are eliminated, along with the need to construct stable cell lines.

How TransAM® transcription factor ELISAs work

The TransAM format is perfect for assaying transcription factor binding to a consensus-binding site. TransAM Kits contain a 96-stripwell plate to which the consensus-binding site oligo has been immobilized. Activated nuclear extract is added to each well and the transcription factor of interest binds specifically to this bound oligonucleotide. A primary antibody specific for an epitope on the bound and active form of the transcription factor is then added followed by subsequent incubation with secondary antibody and Developing Solution to provide an easily quantified, sensitive colorimetric readout (Figure 1).

Figure 1: Flow chart of the TransAM process.

Activated transcription factor in the cell extract binds to the consensus-binding site on the oligo immobilized in the well. Incubation with the supplied primary and secondary antibodies specifically quantifies the amount of activated transcription factor.

* Technology covered under EAT-filed patents and licensed to Active Motif. Use of TransAM in NFκB-related drug discovery may be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA, USA).